EP2843052A1 - Zelldurchlässiges Fusionsprotein zur Erleichterung der Umprogrammierungsinduktion und Verwendung davon - Google Patents

Zelldurchlässiges Fusionsprotein zur Erleichterung der Umprogrammierungsinduktion und Verwendung davon Download PDF

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EP2843052A1
EP2843052A1 EP14182568.7A EP14182568A EP2843052A1 EP 2843052 A1 EP2843052 A1 EP 2843052A1 EP 14182568 A EP14182568 A EP 14182568A EP 2843052 A1 EP2843052 A1 EP 2843052A1
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cell
reprogramming
fusion protein
protein
stem cell
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French (fr)
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EP2843052B1 (de
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Yoon Jeong Park
Gene Lee
Hyun Nam
Jin Sook Suh
Chong-Pyoung Chung
Jue-Yeon Lee
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Nano Intelligent Biomedical Engineering Corp
SNU R&DB Foundation
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Seoul National University R&DB Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/85—Fusion polypeptide containing an RNA binding domain
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/604—Klf-4
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/608—Lin28
    • C—CHEMISTRY; METALLURGY
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1352—Mesenchymal stem cells
    • C12N2502/1364—Dental pulp stem cells, dental follicle stem cells
    • C—CHEMISTRY; METALLURGY
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1361—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from dental pulp or dental follicle stem cells

Definitions

  • the present invention relates to a method of preparing a reprogramming induced pluripotent stem cell from a human-derived somatic cell using a fusion protein in which a reprogramming inducing factor and cell permeable peptide (CPP) are fused, and a fusion protein in which a reprogramming inducing factor and a cell permeable peptide are fused.
  • CPP cell permeable peptide
  • Stem cells have different differentiation potency depending on origins of each stem cell, unlike general somatic cells.
  • an embryonic stem cell is formed from a blastocyst which is one of the earliest stage of embryonic development, and the most important characteristic of the embryonic stem cell is pluripotent. That is, the embryonic stem cell may be differentiated into most types of cells present in the body including three germ layers of endoderm, mesoderm and ectoderm and regenerated.
  • the main weakness in the embryonic stem cell is that a technology of differentiation into a desired cell is not sufficient, and there is a possibility of forming cancer at the time of transplantation. Due to the problem above, there is a limitation in the development of a cellular therapeutic agent. Nevertheless, research into a technology of developing a therapeutic agent utilizing embryonic stem cells in response to shortage of transplant organs or treatment of genetic diseases and nonrenewable diseases has been continuously conducted.
  • Yamanaka group established a reprogramming pluripotent stem cell having similar properties to an embryonic stem cell from a fibroblast of a tail of a mouse by over-expressing four reprogramming transcription factors ( Yamanaka, S, Cell, 126:663-76, 2006 ).
  • Yamanaka group and James A. Thomson group reported that a reprogramming pluripotent stem cell similar to the embryonic stem cell may be formed from a human somatic cell.
  • the two groups used different transcription factors for inducing reprogramming, wherein the Yamanaka group used Oct-4/Sox-2/c-Myc/Klf-4 and Thomson group used Oct-4/Nanog/Sox-2/Lin28 ( Cell.
  • a gene has various problems in that it is not easy to transfer the gene into a cell, in particular, it is significantly difficult to be permeated into a nucleus, a duration in which protein is expressed in a cell is short, and it is significantly difficult to artificially adjust the amount of protein to be expressed in a target cell ( Verma, I.M. and Somia, N., Nature 389:239-242, 1997 ).
  • an introgression method into a cell by an electrode disturbance by applying an electrical stimulation to a cell membrane there is an introgression method into a cell by an electrode disturbance by applying an electrical stimulation to a cell membrane.
  • the introgression method has a disadvantage in that the number of cells which are stuck to each other due to the damage of the cell membrane and do not grow again corresponds to about 70%.
  • the most commonly used method is a method in which a molecule having a gene surrounded with a lipid membrane as a liposome form is introduced into a cell by endocytosis.
  • cytotoxicity is not largely caused as compared to an electrical stimulation method.
  • this experimental method is not the best method, either.
  • the cell permeable method there is a method of transferring a gene by a virus.
  • This method is also generally used when inducing over-expressing materials having a large molecular weight such as a therapeutic agent or a gene in a biological experiment in a cell.
  • this method is an experimental method based on viruses, an introgression efficiency is significantly excellent; meanwhile, at the time of being applied to a clinical testing, there is a limitation in that safety may not be secured. Therefore, a method of safer and effectively delivering materials having biological activity into a cytoplasm or a nucleus of a living cell has been demanded.
  • TAT transactivator of transcription
  • a transactivator of transcription (TAT) protein which is a kind of a human immunodeficiency virus type-1 protein has been substantially studied and it is known that TAT protein effectively passes through a cell membrane and is easily transferred into the cytoplasm. It is known that this function is provided due to property of a protein transduction domain, which is the middle domain of the TAT protein ( Green, M. and Loewenstein, P.M., Cell, 55:1179, 1988 ; Ma, M. and Nath, A., J. Virol., 71:2495, 1997 ).
  • the TAT domain functions directly on the lipid double-layer of the cell membrane ( Vives, E. et al., J. Biol. Chem. 272:16010, 1997 ).
  • the CPP is a protein derived from the virus, which is concerned about toxicity.
  • an antennapedia (ANTP) protein derived from a vinegar fly (drosophila sp.) ( Schwarze, S.R.et al., Trends. Pharmacol. Sci., 21:45, 2000 ) and an artificial peptide in which electrically positive amino acids are listed, cell permeability thereof was confirmed by experiments ( Laus, R. et al., Nature Biotechnol., 18:1269, 2000 ).
  • the present inventors made an effort to develop a more effective method of preparing a reprogramming induced pluripotent stem cell, and found that in the case of directly delivering a cell permeable fusion protein to a target somatic cell instead of inducing the reprogramming of the target somatic cell through the delivery of the genes, the reprogramming of the somatic cell could be induced without genetic modification, such that an induced pluripotent stem cell in which stability of the gene is maintained was capable of being prepared, thereby completing the present invention.
  • An object of the present invention is to provide a cell permeable fusion protein for effectively preparing a reprogramming induced pluripotent stem cell.
  • Another object of the present invention is to provide a method for effectively preparing an induced pluripotent stem cell using the cell permeable fusion protein.
  • the present invention provides a fusion protein for preparing an induced pluripotent stem cell in which a reprogramming inducing factor and a cell permeable peptide are fused.
  • the present invention provides a recombinant polynucleotide encoding the fusion protein for preparing an induced pluripotent stem cell.
  • the present invention provides a recombinant vector containing the recombinant polynucleotide and a recombinant microorganism transformed with the recombinant vector.
  • the present invention provides a method for preparing an induced pluripotent stem cell comprising the steps of: (a) culturing an animal-derived somatic cell in a medium containing the fusion protein for preparing an induced pluripotent stem cell, thereby inducing reprogramming of the somatic cell; and (b) recovering an induced pluripotent stem cell in which reprogramming of the somatic cell is induced.
  • the induced pluripotent stem cell having high efficiency and high stability can be prepared by maximizing the effect of the reprogramming inducing transcription factor beyond the existing viral peptide transporter, in inducing the reprogramming of the somatic cell.
  • the present invention provides a fusion protein for preparing an induced pluripotent stem cell in which a reprogramming inducing factor and a cell permeable peptide are fused, a recombinant polynucleotide encoding the recombinant fusion protein, a recombinant vector containing the recombinant polynucleotide, and a recombinant microorganism transformed with the recombinant vector.
  • a fusion protein in which a reprogramming inducing factor is fused with the specific fusion protein, that is, a cell permeable peptide sequence was prepared without delivering the gene into the somatic cell. Then, it was confirmed that in the case of adding the fusion protein to a culture medium and culturing a somatic cell, the protein was effectively introduced into the somatic cell, and the reprogramming was induced.
  • the reprogramming inducing factor in the present invention is one or more proteins selected from the group consisting of c-Myc, Lin28, Sox2, Klf4 and Oct4.
  • the cell permeable peptide is a peptide having 70 to 80% of an amino acid selected from the group consisting of arginine, lysine and histidine.
  • the cell permeable peptide is one or more selected from the group consisting of a low molecular weight protamine (LMWP) (VSRRRRRRGGRRRR: SEQ ID NO: 2), TAT (YGRKKRRQRRR: SEQ ID NO: 3), penetratin (YGRKKRRQRRR: SEQ ID NO: 4), antennapedia (ANTP: SEQ ID NO: 5), heparin binding domain (HBD) (SSRKKNPNCRRH: SEQ ID NO: 6), D-arginine oligopeptide (R 8 or more), and L-arginine oligopeptide (R 8 or more).
  • LMWP low molecular weight protamine
  • HMD heparin binding domain
  • SSRKKNPNCRRH SEQ ID NO: 6
  • the present invention provides a CPP-reprogramming inducing factor expression vector capable of over-expressing and easily purifying a CPP-reprogramming inducing factor fusion protein.
  • the expression vectors (pCPP-c-Myc, pCPP-Lin28, pCPP-Sox2, pCPP-Klf4, pCPP-Oct4) included 14 amino acids of human c-Myc, Lin28, Sox2, Klf4, Oct4 and CPP transduction domains (in the present drawings, LMWP was used as CPP, and thus, hereinafter, referred to as an LMWP-reprogramming induced fusion protein).
  • the present invention provides the recombinant polynucleotide capable of expressing six histidine residues in terminal of the reprogramming inducing factor (see FIGS. 2 (a) and 2(b) ).
  • a general TA vector, and the like can be used, and the expression of the vector may be performed under control of a T7 promoter and a LacO-operator.
  • the LMWP-reprogramming induced fusion protein was over-expressed in E. coli using the expression vector, followed by purification in the denaturalized state by Ni-chelating affinity chromatography.
  • the purification in the denaturalized state means purification after the LMWP-reprogramming induced fusion protein is denaturalized with 4M to 8M urea.
  • 4M urea the induction is not easily performed, and in the case of using more than 8M urea, agglomeration is caused.
  • the present invention provides a method for preparing an induced pluripotent stem cell comprising the steps of: (a) culturing an animal-derived somatic cell in a medium containing the fusion protein for preparing an induced pluripotent stem cell, thereby inducing reprogramming of the somatic cell; and (b) recovering an induced pluripotent stem cell in which reprogramming of the somatic cell is induced.
  • the present invention is characterized by directly or indirectly co-culturing the somatic cell in which reprogramming is induced in the step (a) and the feeder cell.
  • a low molecular weight protamine (LMWP) is used as a cell permeable peptide, and five reprogramming inducing proteins (c-Myc, Lin28, Sox2, Klf4 and Oct4) are fused therewith, thereby preparing five kinds of fusion proteins.
  • LMWP-reprogramming induced fusion protein into cytoplasm and a cell nucleus within a short time (60 minutes) in a fibroblast group including a human dental pulp-derived stem cell cultured after a human wisdom tooth was extracted to secure a dental pulp tissue, and the secured dental pulp tissue was pulverized, and then confirmed by a Western blotting method and a confocal fluorescent laser scanning microscope. Meanwhile, a reprogramming inducing protein in which LMWP is not encoded, as a negative control protein, was not transported to the cell.
  • the somatic cell derived from human includes human skin, hair, fat, and the like, without limitation.
  • Treatment plans of the five fusion proteins include a case of inducing the reprogramming stem cell through direct and indirect co-culture using the feeder cell and a case of inducing the reprogramming stem cell in a culture dish coated with Matrigel without the feeder cell.
  • the case of using the feeder cell and the direct co-culture means that the five fusion protein is treated four times at intervals of five days and then transported to the feeder cell.
  • the case of using the feeder cell and the indirect co-culture means that the cell is cultured in the culture dish coated with Matrigel and then the feeder cell is cultured onto the insert.
  • the five fusion proteins are treated at intervals of five days to induce the reprogramming.
  • the cell is cultured in the culture dish coated with Matrigel and the five fusion proteins are treated at intervals of five days to induce the reprogramming ( FIG. 7 ).
  • the induced reprogramming stem cell is grown while forming colonies and has a form of a typical embryonic stem cell ( FIG. 8(a) ).
  • Tra-1-60 and SSEA-4 were stained, which means that a complete reprogramming rather than a partial reprogramming was achieved ( FIG. 8(b) ).
  • the medium is a general medium for cell culture containing the fusion protein.
  • a medium in which Knock-out Serum Replacement, Non-Essential Amino Acid, basic FGF, penicillin and streptomycin are supplemented in DMEM/F12 may be used or commercially available mTeSR may be used for culture.
  • the cell permeable reprogramming induced fusion protein according to the present invention may be chemically or biologically fused. However, a biological fusion method is preferred since by-products are less generated.
  • the chemical fusion method is to induce S-S coupling of an amino group of the transcription factor protein and a cell permeable peptide using 1,4-bis-maleimidobutane (BMB), 1,11-bis-maleimidotetraethyleneglycol (BM [PEO]4), 1-ethyl-3-[3-dimethyl aminopropyl] carbodiimide hydrochloride (EDC), succinimidyl-4-[N-maleimidomethylcyclohexane-1-carboxy-[6-amido caproate]] (SMCC) and sulfonides thereof (sulfo-SMCC), succimidyl 6-[3-(2-pyridyldithio)-ropionamido] hexanoate] (SPDP), and
  • the “cell” used in the present invention means a somatic cell or a stem cell having various origins.
  • the “stem cell” is a blast cell having differentiation potency into various kinds of tissues in the body, and may be largely classified into an embryonic stem cell and an adult stem cell.
  • the “embryonic stem cell”, which is a blast cell having differentiation potency means a cell having pluripotency which is differentiated into various tissue cells under appropriate conditions in an undifferentiated state, and includes embryoid bodies derived from the embryonic stem cell with a wide range.
  • the “adult stem cell” means a cell having a limited differentiation potency which is not capable of being differentiated into every tissue in the body but being differentiated into each target organ.
  • the “differentiation potency” means a potency in which a portion of the embryo is capable of being differentiated into various kinds of organs or tissues depending on development conditions in the early development of organisms.
  • the “dedifferentiation” in the present invention means an epigenetic regression process in which a partially or finally differentiated cell is allowed to return to the undifferentiated state such as a pluripotent state or a versatile state, such that new differentiated tissue may be formed.
  • the dedifferentiation phenomenon is possible since it is a reversible process in which epigenetic changes of a cell genome are not fixed but may be erased and may be formed again.
  • the dedifferentiation may be called as "reprogramming” and is directed to a process of changing genetic and expressive profile of the partially or finally differentiated cell so as to be similar to that of the embryonic stem cell.
  • the change includes a change in methylation pattern, a change in an expression rate of a stem cellular gene, and the like.
  • the "induced pluripotent stem cell” used in the present invention means a stem cell having differentiation potency into all triploblastic cells, including the mesoderm, the endoderm and the ectoderm required for forming biological entities, which indicates ultimate potency of the stem cell.
  • the pluripotent stem cellular gene means a marker having potency or a gene having potency of inducing the pluripotency, such that the pluripotent stem cellular gene is classified as a reprogramming inducing factor.
  • the "feeder cell” or the “feeder layer” used in the present invention which is a cell used for culturing the embryonic stem cell or the reprogramming stem cell, is treated with Mitomycin C or is used in a state in which cell division is stopped by irradiation.
  • a mouse embryonic fibroblast (MEF) is usually used and commercially available STO cell line, SNL, or the like, is used.
  • STO cell line SNL, or the like
  • LMWP low molecular weight protamine
  • the gene of the reprogramming inducing factor used as the fusion protein was shown in Table 1, and base sequence and amino acid sequence of the cell permeable peptide, that is, LMWP were shown in Table 2.
  • the requested plasmids were transformed into each competent cell (Novagen, BL21 strain, Rosetta 2(DE3)), the transformed E. coli BL21 (DE3) were selected, colonies were inoculated into 200ml medium, and 1mM of Isopropyl-1-thio- ⁇ -D-galactopyranoside (hereinafter, referred to as IPTG; Duchefa, Netherlands) was added to the medium, thereby obtaining a recombinant LMWP-reprogramming factor, followed by over-expression.
  • IPTG Isopropyl-1-thio- ⁇ -D-galactopyranoside
  • FIG. 3(a) is a photograph of a control protein stained with Coomassie blue
  • FIG. 3(b) is a stained photograph of the LMWP-reprogramming inducing factor.
  • the fusion protein was determined by a Western blotting method.
  • the protein was transferred into a nitrocellulose membrane (NC membrane).
  • NC membrane nitrocellulose membrane
  • the NC membrane with the fusion protein transferred thereto was subjected to blocking in 5% skim-milk at room temperature for 1 hour.
  • the NC membrane was washed three times with a TBST solution (prepared by 8.8g of NaCl, 0.2g of KCl, 3g of Tris base, pH 7.4, and 0.05% Tween20 in 1L solution) for 10 minutes each.
  • the washed NC membrane was reacted with primary antibodies.
  • Example 2 In order to increase a purification yield of the fusion protein prepared in Example 1, Example 2 was conducted under denature conditions in which three dimensional structure of the protein was denatured, and 8M urea was put into buffers (Lysis buffer, Washing buffer and Elution buffer) used for purification.
  • the transformed E.coli BL21 was put into an LB medium containing ampicillin, and cultured with stirring at a rate of 180 rpm and a temperature of 37°C. When the bacterial concentration in the culture medium, OD 600 value, was 0.3, IPTG was put into the medium so as to have a concentration of ImM. Then, after culturing for 4 hours, over-expression of the protein was induced.
  • lysis buffer 50mM NaH 2 PO 4 , 300mM NaCl, 10mM imidazole, 8M urea, pH 8.0
  • lysozyme from chicken egg white (Sigma-Aldrich, USA)
  • 10mg complete EDTA-free Protease Inhibitor Cocktail (Roche, UK) were put thereinto, followed by reaction for 20 minutes, and sonication.
  • the reacted product was centrifuged at 4° to obtain a supernatant, followed by purification with affinity chromatography using His tag present in the fusion protein.
  • the cytoplasm and the nucleus of the fusion protein were separated from each other and confirmed by the Western blotting method.
  • Each 1x10 6 of fibroblast groups containing the human dental pulp-derived stem cell were divided into 10cm dish, and incubated overnight in a general medium for stabilizing the cell, for 20 hours. After the incubating overnight for 20 hours, the LMWP-reprogramming inducing factor and the control fusion protein in a concentration of 4 ⁇ g/ml were treated for 1 hour and 30 minutes.
  • the cells treated with the LMWP-reprogramming inducing factor and the control fusion protein were separated into the cytoplasm and the nucleus with NE-PERTM Nuclear and cytoplasmic extraction reagents (Pierce, USA) according to the experiment method of the manufacturer.
  • the lysate separated into the cytoplasm and the nucleus was protein-quantified with Bradford's assay, subjected to electrophoresis in 10% polyacrylamide gel with 120 volts for 4 hours, and transferred to a nitrocellulose membrane with a transfer buffer (12.5mM Tris, 0.1M glycine, pH 8.3) at 310 milliampere (mA) for 2 hours.
  • the membrane was blocked with a blocking solution (5% Nonfat dry milk, in TBS), and a primary antibody solution (c-Myc, Lin28, Sox2; Cell Signaling, USA, Klf4; Abcam, USA, Oct4; Santa Cruz, USA, LaminB, Actin, GAPDH(Glyceraldehyde 3-phosphate dehydrogenase); Santa Cruz, USA) added to the blocking solution so as to have a concentration of 1 ⁇ g/ml was reacted overnight at 4°C.
  • the secondary antibody to each primary antibody was added to the blocking solution at a ratio of 1:2000 and reacted at room temperature for 1 hour.
  • the reacted products were photosensitized with an X-ray film in a dark room using an enhanced chemo-luminal (ECL).
  • ECL enhanced chemo-luminal
  • GAPDH which is a marker of the cytoplasm
  • Lamin B which is a marker of the nucleus
  • results thereof were shown in FIG. 6(a) .
  • FIG. 6(b) it could be confirmed that when observing the amount of the fusion protein fractionized into the cytoplasm fraction and the nucleic fraction and permeated by an antigen-antibody reaction, the fusion protein was introgressed into the nucleus as well as the cytoplasm at each protein concentration treated within a short time (60 minutes). Meanwhile, the negative control protein could not enter into the cell under the same time and the same concentration as the fusion protein.
  • the fusion protein was marked with the fluorescent marker. 1mg/ml of the prepared protein was dissolved into phosphate buffer (pH 8.5), and fluorescein isothicyanate (FITC) dissolved into dimethyl sulfoxide (DMSO) was mixed therewith, wherein the amount of the FITC corresponds to twice as the calculated value of the molar ratio obtained from the molecular weight of the protein.
  • FITC fluorescein isothicyanate
  • DMSO dimethyl sulfoxide
  • the reaction between each 1/10 of the FITC solution and the protein lysate was allowed to be induced at intervals of 10 minutes, followed by shading at 4°C to induce the combination reaction.
  • the product was purified by reverse phase liquid chromatography for purification.
  • 0.1% TFA/H 2 O and 0.092% TFA/acetonitrile were allowed to flow in C 18 column having a diameter of 5 cm, with a change of 0 to 60% at a flow rate of 20ml/min, for 30 minutes.
  • the wavelength of UV detector was set to be 220nm
  • the wavelength of fluorescence detector was as follows: Ex: 493.5nm, Em: 460nm, and the wavelength of UV detector was set to be 220nm.
  • the only pure fluorescent marked fusion protein was fractionized and freeze-dried.
  • LMWP-Reprogramming Inducing Factor Fusion Protein 5x10 4 cells were divided into 4-well chamber, and in MEM medium not containing fetal bovine serum (FBS), each 4 ⁇ g/ml of the fluorescent marked LMWP-reprogramming inducing factor fusion protein prepared by the preparation of Example 3-2 and the negative control protein was injected into each well, then after 1 hour, the product was twice washed with phosphate buffer solution (PBS) and treated with a dye staining the nucleus (Hoechst 33342, blue) at room temperature for 10 minutes.
  • FBS phosphate buffer solution
  • a mouse embryonic fibroblast treated with mitomycin C was cultured in 6-well plate coated with 0.2% gelatin for 24 hours, in DMEM medium containing 10% FBS. The next day, the primarily cultured human dental pulp-derived mesenchyma stem cell in a concentration of 5x10 4 for each well was put thereinto and cultured for three more days. Then, the culture medium was changed every day with 20% Knock-out Serum Replacement, 1% Non-Essential Amino Acid, 100ng/mL basic FGF or mTeSRTM1, in the embryonic stem cell culture medium DMEM/F12.
  • the reprogramming inducing medium contained five LMWP-reprogramming inducing factor fusion proteins in each concentration of 8 ⁇ g/ml with 20% Knock-out Serum Replacement, 1% Non-Essential Amino Acid, 100ng/mL basic FGF or mTeSRTM1 in DMEM/F12, and the reprogramming inducing medium was put thereinto at intervals of five days. Colonies were shown in 20 days and then the embryonic stem cell culture medium was used ( FIG. 7 ). In order to differentiate the complete reprogramming and the partial reprogramming, live-cell imaging was conducted with Tra-1-60 and SSEA-4. When the colonies in which the complete reprogramming was conducted grew to have a size available for subculture, subculture was performed. It could be confirmed from FIG. 8(a) that each colony had a typical shape of the embryonic stem cell, and it could be confirmed from FIG. 8(b) that as staining results with Tra-1-60 and SSEA-4, the complete reprogramming was performed
  • a mouse embryonic fibroblast treated with mitomycin C was cultured on the insert in DMEM medium containing 10% FBS for 3 days.
  • the human dental deciduous pulp-derived mesenchyma stem cell was cultured in a concentration of 5x10 4 for each well in the culture dish coated with Matrigel, and after 3 days, the culture medium was changed into the reprogramming inducing medium.
  • the reprogramming inducing medium contained five LMWP-reprogramming inducing factor fusion proteins in each concentration of 8 ⁇ g/ml with 20% Knock-out Serum Replacement, 1% Non-Essential Amino Acid, 100ng/mL basic FGF or mTeSRTM1 in DMEM/F12, and the reprogramming inducing medium was put thereinto at intervals of five days. Then, on days in which the reprogramming inducing medium was not put thereinto, the culture medium was changed every day with 20% Knock-out Serum Replacement, 1% Non-Essential Amino Acid, 100ng/mL basic FGF or mTeSRTM1, in the embryonic stem cell culture medium DMEM/F12.
  • Colonies were shown in 20 days and then the embryonic stem cell culture medium was used. When the colonies grew to have a size available for subculture, subculture was performed. Then, in order to differentiate the complete reprogramming and the partial reprogramming, live-cell imaging was conducted with Tra-1-60 and SSEA-4 ( FIG. 7 ). When the colonies in which the complete reprogramming was conducted grew to have a size available for subculture, subculture was performed. It could be confirmed from FIG. 8(a) that each colony had a typical shape of the embryonic stem cell, and it could be confirmed from FIG. 8(b) that as staining results with Tra-1-60 and SSEA-4, the complete reprogramming was performed.
  • the primarily cultured human dental pulp-derived mesenchyma stem cell was put into a culture dish coated with Matrigel with a concentration of 5x10 4 for each well and cultured for three days. After 3 days, the culture medium was changed into the reprogramming inducing medium.
  • the reprogramming inducing medium contained five LMWP-reprogramming inducing factor fusion proteins in each concentration of 8 ⁇ g/ml with 20% Knock-out Serum Replacement, 1% Non-Essential Amino Acid, 100ng/mL basic FGF or mTeSRTM1 in DMEM/F12, and the reprogramming inducing medium was put thereinto at intervals of five days.
  • the culture medium was changed every day with 20% Knock-out Serum Replacement, 1% Non-Essential Amino Acid, 100ng/mL basic FGF or mTeSRTM1, in the embryonic stem cell culture medium DMEM/F12. Colonies were shown in 20 days and then the embryonic stem cell culture medium was used. When the colonies grew to have a size available for subculture, subculture was performed. Then, in order to differentiate the complete reprogramming and the partial reprogramming, live-cell imaging was conducted with Tra-1-60 and SSEA-4 ( FIG. 7 ).

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